The formation of myelin by Schwann cells requires the precise timing of expression and the exact positioning of a number of specific proteins. How these proteins are delivered to the correct location and what specific functions they have is unknown. The major peripheral myelin protein, Po, is thought to interact with itself (hemophilic binding) possibly via its carbohydrate residues and thus play a role in compaction of myelin. Moreover, changes in the glycosylation of Po have been associated with a changes in its expression, although evidence for a direct correlation is lacking. The overall goals of this proposal are to determine whether the oligosaccharide chain of Po is involved in hemophilic interaction and whether changes in glycosylation constitute the key mechanism for regulation Po expression and sorting in the Schwann cell. As an abundance of Po is required for these studies, the immediate goals are to over-express the glycoprotein by gene amplification using the dihydrofolate reductase (DHFR) strategy in cells transfected with the cDNA for Po. For the adhesion studies, cells that have no endogenous myelin proteins, Chinese hamster ovary (CHO) cells, will be used which will assist in the accurate interpretation of the data. Through specific manipulation of the sugar structure, assessment of its contribution of adhesion will be made. Studies on the expression/sorting of Po will be carried out in Schwann cells. However, since in Schwann cells the transfected Po must be distinguished from the endogenous glycoprotein, a "reporter" group of 7-10 amino acids will be attached to the C-terminal portion of the transfected glycoprotein. To ensure that Po with and without the reporter peptide is expressed/sorted in an identifical manner, both will be characterized in CHO cells, prior to the studies with Schwann cells. Eventually, the expression of Po-reporter will be monitored in Schwann cells in relation to the addition of axolemma and/or extracellular matrix to the culture, before and after treatment with specific inhibitors of glycosylation. Finally the long term goals of this study are to apply the expertise gained in this study ot other specific myelin proteins and hence to gain an understanding of myelin-ation in the peripheral nervous system. Such information is critical in understanding the basic mechanism and pathology of any peripheral neuropathy which involves demyelination and remyelination e.g., Charcot-Marie-Tooth disease.